Lignocellulosic materials contain carbohydrate in the form of complex polymers. The polymers can be broken down to simple sugars which in turn can be used to produce various products including ethanol. Various biomass feedstocks have a lignocellulosic structure where lignin surrounds the hemicelluloses and cellulosic carbohydrates whose release increases upon enzymatic treatment with cellulases. However, enzyme cost and low efficiency of hydrolysis are major issues associated with lignocellulosic materials' potential as a source of fermentable sugars.
Beta-glucosidase (BG) is a key cellulosic enzyme that hydrolyzes the di-saccharide cellobiose into the mono-saccharide glucose. This last step in glucan saccharification generates the preferred sugar (glucose) for fermentation, and effectively clears the enzymatic pathway of its end product. If glucose concentration is high, end-product inhibition of upstream cellulases occurs. End-product inhibition decreases flux through the saccharification pathway by decreasing the catalytic activity of upstream enzymes including cellobiohydroblases (CBH1, CBH2) and endoglucanase (EG). Thus, overall saccharification efficiency can be diminished in glucose titer and kinetics of sugar production.
Available BG enzymes can have reduced activity in the presence of glucose levels typically found in biomass saccharification reactions, i.e. product inhibition. Reduced activity generally can be quantified as the amount of substrate hydrolysis that occurs in a specified time under specified reaction conditions in the presence of glucose vs. in the absence of glucose. Product inhibition could result in altered kinetic properties of enzymes, such as Michaelis-Menten constant (Km) and maximum velocity (Vmax), depending on different inhibition mechanisms. Competitive inhibition increases the apparent Km without affecting the value of Vmax. Noncompetitive inhibition decreases the apparent Vmax without affecting the Km. Uncompetitive inhibition decreases the apparent values of both Km and Vmax. Mixed inhibition also exists and has more complex effects on kinetic properties. These BG enzymes may also have sub-optimal half-lives and thermo-tolerance, making it useful to have an improved form of BG to get the highest glucose yields during saccharification. The present disclosure provides, among other things, improved beta-glucosidase enzymes that increase the conversion of cellobiose to glucose, and thus improve saccharification efficiency.